Project 1 is a proposal to study structure, assembly, and disassembly of two intricate molecular machines: clathrin coated vesicles and components of the S. cerevisiae kinetochore. (1) Clathrin coated vesicles carry membrane traffic to endosomes from the plasma membrane and the trans-Golgi network. The coats of these structures, lattices of clathrin, organize membrane vesiculation. The point of departure for the proposed work is the molecular structure of clathrin coats, determined during the previous grant period by fitting a7.9A resolution image reconstruction from single-particle cryoEM with atomic models derived from x-ray crystallography. This effort was a collaboration among three of the four P.I.'s of the present proposal. Our aims are now: (i) to extend further the resolution of the cryoEM map and to analyze designs of additional clathrin lattices;(ii)to determine the structural mechanism of clathrin disassembly ("uncoating");(iii)to study individual coated vesicles by cryo-electron tomography. (2) Kinetochores are the multiprotein complexes that assemble on centromeric DNA and mediate attachment to mitotic spindle microtubules. To understand how kinetochore assembly determines faithful segregation of chromosomes requires analysis of kinetochore structure. Kinetochores of budding yeast contain over 60 distinct protein species, organized into over 14 subcomplexes. We have begun to study the structures of some of these subcomplexes during the previous grant period. We now propose more detailed analysis of the DASH/Daml complex, using cryo- electron tomography. DASH, a heterodecameric complex of ten distinct proteins, assembles into rings that encircle microtubules. Relevance: Clathrin-mediated pathways include endocytosis of transferrin, LDL, and viruses, and reuptake of membrane after release of neurotransmitter at synapses, all processes relevant to human disease. Kinetochores are critical for chromosome segregation into daughter cells;errors in segregation can lead to cancer.